CN108060112A - One plant of bacteria cellulose production bacterial strain and its construction method and application - Google Patents
One plant of bacteria cellulose production bacterial strain and its construction method and application Download PDFInfo
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- CN108060112A CN108060112A CN201711210409.6A CN201711210409A CN108060112A CN 108060112 A CN108060112 A CN 108060112A CN 201711210409 A CN201711210409 A CN 201711210409A CN 108060112 A CN108060112 A CN 108060112A
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- bacterial strain
- bacteria cellulose
- cellulose
- acetobacter xylinum
- bcsb
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- 241000894006 Bacteria Species 0.000 title claims abstract description 58
- 229920002678 cellulose Polymers 0.000 title claims abstract description 49
- 239000001913 cellulose Substances 0.000 title claims abstract description 48
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 238000010276 construction Methods 0.000 title claims abstract description 11
- 235000002837 Acetobacter xylinum Nutrition 0.000 claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 101150082227 bcsB gene Proteins 0.000 claims abstract description 11
- 108010040093 cellulose synthase Proteins 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 241001136169 Komagataeibacter xylinus Species 0.000 claims abstract 7
- 239000013612 plasmid Substances 0.000 claims description 16
- 239000013613 expression plasmid Substances 0.000 claims description 4
- 238000005215 recombination Methods 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims description 2
- 235000010980 cellulose Nutrition 0.000 abstract 4
- 244000235858 Acetobacter xylinum Species 0.000 description 23
- 239000002609 medium Substances 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000035784 germination Effects 0.000 description 6
- 238000012795 verification Methods 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 5
- 230000005611 electricity Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 241000589180 Rhizobium Species 0.000 description 2
- 241000192023 Sarcina Species 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 241000589220 Acetobacter Species 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 101150110946 gatC gene Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008104 plant cellulose Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
- C12N9/1059—Cellulose synthases (2.4.1.12; 2.4.1.29)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Biomedical Technology (AREA)
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- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses one plant of bacteria celluloses to produce bacterial strain, the bcsB subunits being overexpressed in acetobacter xylinum in cellulose synthase, the bcsB subunits in the cellulose synthase, gene order such as SEQ ID NO:Shown in 1.Construction method the invention also discloses above-mentioned bacteria cellulose production bacterial strain and its application in bacteria cellulose is prepared.The bacteria cellulose output that the present invention obtains recombinant bacterial strain reaches as high as 5g/L, the whole output increased more than 20% compared with original strain, while by half that fermentation time reduction is original strain.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one plant of bacteria cellulose production bacterial strain and its construction method
With application.
Background technology
Bacteria cellulose (Bacterical cellulose, BC) refers under different conditions, by acetic acid Pseudomonas
(Acetobacter), Agrobacterium (Agrobacterium), rhizobium (Rhizobium) and Sarcina
(Sarcina) general designation of the cellulose of certain Microbe synthesis in such as.Bacteria cellulose is compared with plant cellulose, without wood
Quality and hemicellulose impurities, thus before commercial Application, numerous and diverse pretreatment need not be carried out, extraction process is simple；Except this
Outside, there is bacteria cellulose fine network structure, higher mechanical strength, higher water suction and water retention property, synthesis to be
The property of many uniquenesses such as Modulatory character, good biocompatibility and biodegradability, thus be considered as performance it is best,
The highest cellulose of use value, still, in current production process, the speed of production of acetobacter xylinum is slow, yield lower limit
Its extensive use, therefore, the yield and yield for improving bacteria cellulose are highly desirable.
Acetobacter xylinum is found and studies earliest most thorough cellulose producing bacterial strain, is the conjunction being currently known
The bacterial strain most strong into cellulose ability.At present, the selection and breeding of the bacterial strain of domestic production bacteria cellulose mainly divide from natural surroundings
From cellulose producing strains, strain excellent is then selected by traditional acclimation method；Foreign countries using traditional domestication except being selected
Outside educating, gene engineering method is additionally used, improvement obtains strain excellent.
The content of the invention
The technical problem to be solved in the invention is to provide one plant of bacteria cellulose production bacterial strain, to solve the prior art
The problem of middle bacteria cellulose production producing strain is low, fermentation time is long.
The present invention also technical problems to be solved are to provide the construction method of above-mentioned bacteria cellulose production bacterial strain.
Last technical problems to be solved of the invention are to provide the application of above-mentioned bacteria cellulose production bacterial strain.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
One plant of bacteria cellulose produces bacterial strain, the bcsB subunits being overexpressed in acetobacter xylinum in cellulose synthase, wherein
BcsB subunits are primarily involved in the synthesis of bacteria cellulose.
Preferably, the acetobacter xylinum is acetobacter xylinum ATCC700178.
Preferably, the bcsB subunits in the cellulose synthase, gene order such as SEQ ID NO:Shown in 1.
The construction method of above-mentioned bacteria cellulose production bacterial strain, includes the following steps：
(1) gene order of the bcsB subunits in cellulose synthase is cloned into expression plasmid, obtains recombinant plasmid；
(2) by recombinant plasmid transformed acetobacter xylinum in step (1), acetobacter xylinum gene recombination bacterium had both been obtained.
In step (1), the expression plasmid is pSA-19.
In step (2), the acetobacter xylinum acetobacter xylinum ATCC700178.
Above-mentioned bacteria cellulose production bacterial strain in bacteria cellulose is produced in fermentation apply protection scope of the present invention it
It is interior.
Advantageous effect：
The present invention carried out table by that will synthesize relevant cellulose synthase subunit with bacteria cellulose in acetobacter xylinum
Up to the yield that the bacterial strain bacteria cellulose is improved with this, the bacteria cellulose output of obtained bacterial strain reaches as high as 5g/L, with original
Beginning bacterial strain compares whole output increased more than 20%, while is the half of original strain by fermentation time reduction.
Description of the drawings
Fig. 1 bacterium colonies PCR verifies electrophoretogram.
Fig. 2 goes out bacterium germination and ferments to obtain the dry weight of bacteria cellulose and yield comparison diagram with recombinant bacterium, and 0 is bacterium germination,
700178-B is the recombinant bacterium that the present invention is built.
Fig. 3 goes out bacterium germination to scheme with recombinant bacterium sugar consumption velocity contrast, and 0 is bacterium germination, and 700178-B is the weight that the present invention is built
Group bacterium.
Fig. 4 goes out bacterium germination, and growth obtains the comparison diagram of cellulose dry weight at any time with recombinant bacterium fermentation, and 0 is bacterium germination,
700178-B is the recombinant bacterium that the present invention is built.
Specific embodiment
According to following embodiments, the present invention may be better understood.It is however, as it will be easily appreciated by one skilled in the art that real
It applies the described content of example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.
Embodiment 1：The clone of acetobacter xylinum cellulose synthase subunit bcsB genes
Expanded firstly for relevant subunit (bcsB) is secreted with bacteria cellulose in the cellulose synthase of acetobacter xylinum
Increasing and sequencing, the DNA fragmentation of 470bp is obtained by amplification.Detailed process is as follows：
First, the genome of acetobacter xylinum ATCC700178 is extracted, designs primer pair bcsB-FA, bcsB-RC, with extraction
Acetobacter xylinum ATCC 700178 genome for template carry out PCR amplification.PCR reaction systems are 5 × PrimeSTAR
buffer(Mg2+)20.0μl、dNTP Mixture(2mM)10.0μl、bcsB-FA1.0μl、 bcsB-RC 1.0μl、
1.0 μ l of PrimeSTAR, 0.5 μ l of template；100.0 μ l are supplemented to sterile water, are then distributed into 25.0 μ l/ pipes.Reaction
Condition：95℃5min；95℃10sec；60℃30sec；72℃3minc；72℃10min；35 Xun Huans, the PCR expanded
Product passes through Gel Extraction kit.
Primer needed for above-mentioned is as follows：
bcsB-FA：tatgaccatgattacgaattcgatatgaaaatggtgtccctgatcgcg；
bcsB-RC：cttgcatgcctgcaggtcgacgatcacgttctctgcctttcttcctgc.
Embodiment 2：The structure of recombinant plasmid pSA19-bcsB and verification.
According to acetobacter xylinum endogenous plasmid sequent synthesis plasmid fragments pAH4 on NCBI, by plasmid pAH4 and plasmid
PUC18 obtains the recombinant plasmid pSA19 with ammonia benzyl resistance with being recombinated after hindIII single endonuclease digestions.With EcoRI and SalI double digestions
PSA19 plasmids, gel recycling.The target fragment that above-mentioned glue recycles is connected on pSA19 plasmids by one-step cloning, from
And construct the carrier pSA19-bcsB using ammonia benzyl resistance as selected marker.Then verified for pSA19-bcsB carriers,
Double digestion verification is carried out, verification result is consistent with expection, send double digestion result plasmid in line to sequencing, with expected phase
Symbol, it was demonstrated that vector construction success.
Embodiment 3：PSA19-bcsB carriers turn the structure and Molecular of 700178 transformant of acetobacter xylinum.
The pSA19-bcsB carriers of 1 μ g are converted into acetobacter xylinum ATCC700178, conversion process uses electrotransformation.Containing
Have and transformant is screened on the Selective agar medium of ammonia benzyl resistance, the acetobacter xylinum bcsB for finally obtaining one plant of inheritance stability turns
Beggar.It is verified through PCR, it was demonstrated that above-mentioned transformant is successively inserted into pSA19-bcsB plasmids.
The specific method of above-mentioned electricity conversion is as follows：
The preparation of competence：
1st, a ring acetobacter xylinum seed is scraped from tablet in the 500ml conical flasks equipped with 100ml seed liquors, is added in simultaneously
The cellulase for crossing film degerming causes the cellulose enzyme amount in every milliliter of culture medium for 0.5U, 30 DEG C, 150rpm, cultivates 18h, makes
The OD values of bacterium solution are obtained between 0.6-0.8.
2nd, obtained bacterium solution 5000rpm will be cultivated, centrifuges 5min, abandoned supernatant and collect thalline, then the EPB solution with 5ml
(sucrose solution of 284mmol and the phosphate buffer of 100mmol, pH7.4 be sterilized separately after with 1:19 ratios are mixed to get)
It washes twice.Finally plus the EPB solution of 3.3ml is resuspended, packing, often 560 μ l of pipe.
The conversion of acetobacter xylinum：
20 μ l (concentration is about 30ng/ μ l) is taken to add in the competence of above-mentioned packing obtained plasmid psa19-bcsB,
It after 10min is placed on ice, is transferred in 2mm electricity revolving cups, adds in electricity in electroporation and turn, electricity turns condition as voltage 3000V, resistance
200 Ω, 25 μ F of capacitance.
Recovery, coated plate：The seed liquor containing cellulase of 1ml is added in the bacterium after electricity converts, then at 30 DEG C,
150rpm, recovery 3h.After the completion of recovery, the 100 above-mentioned bacterium solutions of μ l is taken to be coated on the agar culture of the ammonia benzyl resistance containing 75 μ g/ml
Base, is cultivated 3-4 days, selects the transformant grown on agar medium by 30 DEG C.Simultaneously for the sense of no addition purpose plasmid
It is carried out after the same method by state, is coated on according to identical amount on identical agar medium and is used as competence growing state
Detection and negative control.
Above-mentioned agar medium (i.e. Selective agar medium)：Glucose 20g/l, yeast extract 5g/l, peptone 5g/l, agar
20g/l, 75 μ g/ml of ammonia benzyl antibiotic.
The further picking of transformant grown on tablet will be selected to carry out secondary screening to conversion minimal medium tablet, it will be multiple
The transformant selected on sieve culture medium carries out bacterium colony PCR verifications, and the primer pair of verification is yz-B-1, yz-B-2；
yz-B-1:gagttagctcactcattaggcaccc
yz-B-2:gatcacgttctctgcctttcttcctgc
The gel electrophoresis figure of PCR results is as shown in Figure 1.
Embodiment 4：Recombinant bacterium acetobacter xylinum ATCC700178-bcsB is subjected to fermentation verification.
Acetobacter xylinum transformant and starting strain are activated respectively, are inoculated into seed culture medium, 30 DEG C, 150rpm (shakes
Bed) culture 18-20h after, be inoculated in 100ml (triangular flask of 500ml) fermentation medium, while add in the fermentation medium
1ml absolute ethyl alcohols.While inoculation, Initial sugar concentration is measured by sampling.After 30 DEG C of quiescent culture 6d, rinsed overnight with distilled water
Cellulose membrane is to remove the culture medium of film surface and impurity, then cellulose membrane is immersed in the sodium hydroxide solution of 0.5M and is boiled
Boiling 1h is translucent to remove the thalline of attachment removal to milky, and by treated, cellulose membrane drains on filter paper, the fibre that will be drained
The plain film of dimension dries to constant weight in an oven in 65 DEG C, weighs (i.e. cellulose dry weight), as shown in Figure 2.
Embodiment 5：
Obtained recombinant bacterial strain 700178-bcsB sending and receiving ferment is observed into the yield of the bacterial strain and original strain with residual sugar at any time
Between variation, as shown in Figure 3；Sugar consumptions of the recombinant bacterial strain 700178-B in 0-9d early period declines rapid, corresponding bacteria cellulose
Yield be also substantially increased, and after 9d, sugared concentration declines slow, finally tends towards stability, the yield of corresponding bacteria cellulose
It is basically unchanged；From Yield mapping 4 as can be seen that final in fermentation, the yield of 700178-acsB is improved than original strain
47.8%, 700178-acsB reach maximum production in fermentation 9d, and original strain reaches maximum production in fermentation 14d or so, therefore
Fermentation period shortens 5d.
Sequence table
<110>Nanjing University of Technology
<120>One plant of bacteria cellulose production bacterial strain and its construction method and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2414
<212> DNA
<213>Acetobacter xylinum ATCC700178 (Bacillus xylinus ATCC700178)
<400> 1
gatatgaaaa tggtgtccct gatcgcgctg ctggtctttg caacgggggc acaggctgcg 60
cctgttgctt ccaaggcgcc agctccgcag cccgcaggtt cagacctgcc acctctccct 120
gccgcaccgc cgcaggctgc tccgcccgca gccgcgagtg ccgccccgcc cgccacaacc 180
ccggcggcgg atgcctcagc agccagcgcg gctgatgcgg ttgtggacaa tgccgagaac 240
gccatcgccg ggtctgacgt ggcgacggtg catacatatt ccctcaggga acttggtgcg 300
cagagtgccc tcaaaatgca gggcgctgct acgctgcagg gcctgcagtt cggtattccg 360
gccgaccagc tcgtgacttc ggcgcggctt gtcgtgtcgg gtgcgatgtc gcccagcctc 420
cagcctgaca ccagcgcggt cacgatcacg ctgaacgaac agttcatcgg cacgctgcgg 480
cctgacccca cacaccctac atttgggccg ctttcgtttg atatcaaccc catcttcttc 540
atcagtggca accggctgaa tttcagcttc gcttcaagct cgaagggctg cacggacccc 600
agcaacgggt tgttctgggc cagcgtgtcc gaacattccg agctgcagat caccaccatc 660
ccgcttcccc cgcatcgcca gctgtcgcgt ctgccccagc cgttcttcga caagaacgta 720
aagcagaaga tcgtcattcc gttcgttctc gcacagacat ttgatcccga agtgctgaag 780
gcgacgggca tcctggcatc gtggttcggc cagcagaccg attaccgtgg cgtcaccttc 840
ccggtcttct ccaccattcc gcaaacgggc aacgccgttg ttgtcggcgt ggctgacgag 900
ctgccttccg ccctcgggcg ccaggcggtc agtggcccca cgcttatgga agtggccaat 960
ccatccgacc ccaacggcac gatcctgctc gtaaccgggc gcgaccgtga tgaagtcatc 1020
accgcgagca agggcatcgg ttttggttcg agcaccctgc cgacagccaa ccgcatggac 1080
gtggcgccga tcgaggtcgg ggcccgcgtg gcgaatgacg cgccctcctt cattccgacc 1140
aaccgcccgg tccgcctggg cgaactggtg ccagacagcg ccctgcaggc tgaaggttac 1200
gcccctggcg cgctggcggt gccattccgt gtctcgcctg acctgtatac gtggcgcgat 1260
cggccgaaca agctgaacgt ccgtttccgc gcgccgccgg ggccgatcgt ggatgtgtcg 1320
cgctcgtcgc tcaatgtagg catcaacgat acctatctcg aggcctatcc gctgcgtgag 1380
ccggattcac cgctggacca gctcctgcat ggggtgggcc ttggccatcg taataatgac 1440
agcgtgcagc agcacaccat gcccatcccg acctaccggg tctttggcca gaaccagctg 1500
ctgttctatt tcgagatggc ggcgatggtc gagccgggct gcaaacccgg cccgagcacg 1560
ttccatatgg gcattgatcc caattcgacg atcgatctgt ccaactccta tcacatcacc 1620
cagatgccca acctcgcctt catggccagt gcgggctttc cgttcaccac ctatgccgac 1680
ctgtcgcgct cggccgtggt gctgcccgaa caccccaatg gcatgattgt cagcgcctat 1740
ctcgacctca tgggcttcat gggggcgacg acatggtatc cggtgtctgg cgttgatgtg 1800
gtctccagcg accatgtgaa tgacgtggcg gaccggaacc tgattgtcct gtccacgctg 1860
gccaatagcg gtgatgtttc gcagctgctg agcaattcgg cctatcagat ttccgatggg 1920
cggctgcaca tggccctgcg ttcgacgctg agcggcgtgt ggaacctttt ccaggatccc 1980
atgtcggcca tcaacagcac ggccccgacc gatgtcgaga gcacgctgac cggtggcgtg 2040
gccgcgatgg tcgaggcgga atcgccgctg gcatcgggtc ggaccgttct cgcgctgctt 2100
tcgggtgacg ggcaggggct caacaacctt gtgcagatcc tggcgcagcg gaaaaaccag 2160
gccaagatcc agggtgatct ggtgctggca catggggatg acctgacctc ctaccgcagc 2220
tcgccgctgt atacggttgg caccgtgccg ctgtggctca agcctgactg gtatatgcac 2280
aaccatccca gccgcgtggt cgtggttggc ctgttcggtt gccttctggt ggtggctgtc 2340
ctgatgcgcg ccctgaccaa gcatgctctg cgccgccgtc gggagttgca ggaagaaagg 2400
cagagaacgt gatc 2414
<210> 2
<211> 4002
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aagcttctgg cgggcggatt cctgttcagc ccgcttttgc gcctcgcgtg ttcccgcgcc 60
aatcgctcca ttatggcccg ctgccgtcct gcctctcctg ctccttccgc tcgatggcat 120
cccgacccac ctcgtcggcc agatcggcga accagtccat caggtcttcc gctgcctgct 180
gaaccttggc gatggcctgt tccgcgcttt cgatgatatg gcgcagggct ttcgcaatcc 240
cgttcttctc ggcggcatcc gcatttgccc tggcacggta ggttctgggt tcatactccc 300
gaccttcctg ctcggcctga tgcgcggcct tgcgttcgac cgccgtgctg tgcggcccga 360
gatggacgcc gggcacaacc tcgacaccct gccgctcata actccggtga tcgacgcggg 420
cggtctgtcc ggcccgctct agcagcctgt cgggttgggc attttgcggc tgataacgcc 480
aaggctgacc tggcttatgc tgcctgaagc cgcgccattc ttttacagtt gtatgcgaga 540
gcaacgagtg tccattcggt cgtgactttt gcaaggccac gcaggctgaa ttttctgaag 600
cccatgatgc ttttgataat tccaaagacc ggctccacgg tctgttttcg tcgtctgtaa 660
agatctccgg cttctgtagt ttccagcctg tccttcatgg caagccgcca gggttcggtt 720
atccggcgtg gctccctttc tgcgggccgg ggtcggaagt cgtaaggtct gcgggcacag 780
ggccgtccaa tggcgaccag cggatcaatg cccttttccc gcagtttccg gaccgcctgc 840
ccgctggcgt aaccggtatc ggcgagcact gtctttggga gaccgattgt gtcttccatc 900
gacagcaccg tgtcggcaaa ggacggcgca tccgctgatg tggcgacaac gtcggttgtc 960
acgatcaact ggctgccttc ggcgcacacc acggcctggg cattgtaagc ctgccggaac 1020
tcgtgggcgt ccgaacgccg catgaggcgg ctgtcgggat cggtcagact gatctgtcgg 1080
tcgggtggtg gttcatcact cgggcggttt gggcgcccgg ccgcgacgcc ctgttttcgc 1140
atcataagcg gctttcttct tctcgtaggc cggtcgcgcc gtttcagcct gcgccttcgc 1200
atcagcttcc agccgggcgc aggcttcgtc agcttctttc agcgtttccc gccgggcaag 1260
ctcttccggc aatgctgcgg atctctgtct gtgacgtccg catctccgcc tggtccatca 1320
gtttcgcgat atccacagcc agctgttcgc gcacgcctga tccggtcgta gcgcaccgaa 1380
cggtatttcg atgcgtcagc atcgattttc gtgccgtcga tcgacaccac gcccagacgc 1440
agcagacccg tctcgcgcgc cagaagcagg acctgcgcaa atgcagcttc aatggctgtc 1500
cggttcgtcc ggcggaaggt cgcaatcgta tcatgatccg gatgcaggtt cgccgccacg 1560
aatcgcaccc cgatgtcgcg atatgtcgcc cgctcgatcc ggcgtgagga aaacaacccg 1620
ttcgcatagc tgaagatcag aagggccagc atcaggcgcg gatgatactg cgccttgcct 1680
cccgtgcgca ctggcacgca gaacgcactc atcggaaccc gctcaacggc ggctacaatg 1740
aaatgcgcca tatcatcagc aggaagccac gacttcagat caggcggcag aagatacggc 1800
tgagaccggt caaacgggat gaagctgctc atcacaccac cttacaatcg cccccttcac 1860
agggtacccc aacccgacag gctgctagaa ccgccgtaga agcgtccgag gcattcgcag 1920
tgtcgaaacc ccgcctaatg atctggacgg ccctctgtgc cttcctgctg gtctctggcg 1980
ggtggttggc agcgttctgg gtaggcagac acgatggctg ggccgctggt caggtcgatg 2040
gcagacagga agccctcacc gccaatgccg ccgcgtcatg ggcgaatacc accagcggga 2100
agatggccaa gcaactggat gacctcggca accttcaacc tttggcgact tgcaacgtcc 2160
ccggattctc catccagaag ggggaaaagg gtgttcgctg gtgtgtggtt gctggaacag 2220
acgggcagtt ccacgggtgg gcgatgccct gacgaaccct tccctcttcc tgagcaattc 2280
ggaagatcaa tttcctctag cctaacacgt cgaaaacggg agttttccac caaaaaagag 2340
agacctacag agagattaaa tttctttctc tttcttaacc atagtcaacc cgcgcgagac 2400
tgcggaaaaa tgcttgtaat aggttacagg atatgtaacc cagaagttac aggggctgta 2460
acctattagc ccgttatcaa caggggtgcg agatgtcccg gattgtcaga ctgaccacca 2520
agcggcaaat ggccgaccag caggccgcag ctaccattgc cgaacaactg gaactcatca 2580
ctccggaaat gctggaaggc gctccgggag acctgaaact gttgctgtca cgggctatct 2640
acagcgcaca aaagcaatcg cgcccgaaca ccgaaggact ttggccggga gtttcaccat 2700
gattagccgc gaccagacga aacttgtgtg ggatgccatc cgcgcccttc cgccagaaga 2760
tcgcccccag caggtacgtc acgccttcga tctggccttg ctgtcactgc gacaggatac 2820
cggcgaaatc atgatgcgcc gtgatgaact tgccgaagaa atcggctgtt ctccgcagaa 2880
cgtcagccaa attatgggcg ttctcgagcg tatgggtgcc gtccgtcgaa cccgccaaaa 2940
ggtgccgggg atcagaggac cgggtgtggc aatatattac atcaacccgc atgtcggctg 3000
gaatggctct ctagatgctc gcaaggcaca ggctgaagaa atccatccgc cggtacagct 3060
tgagcttctg caagggggag ccaaatgagt tcccgtagaa gcagtagagc acagggcacc 3120
aatccaaaag ctctaggatt aaaccctaga gcacttggat taagtcctaa acaattagga 3180
attagccccc gtcagctcgg gattagccct aagcaactcg caaaaaagag gcaaatcatg 3240
accgacctat ccgacgaact ggccgccaaa cgggcggcaa tccgtgcagc ccgcgaatgc 3300
acagaaccgt cgctgtctgc ggcggaggct atcgccttgc tggaatccga tctggttatg 3360
gttcaggcag ctatcgacgc tctacacgcc gaggaacgcc gtgcaggttg agtggtcgaa 3420
gctggcccgt tctgatgcgg aagcaatcag agcctacctg ttggatcgaa acccatacgc 3480
agctaagcga attttactcc gtctgatcga tgcgacaaaa gacttggcaa tgttcccgag 3540
catcggtcgg atagggctgg acggcacccg cgaatgggtc gtcgcccagc cctacgttct 3600
gctctacgaa gtcaatgaaa tggccggaat cgttaaaatc ctgcgtgttt ggcacagcgc 3660
ccaagaccgc tgaatagcct ctaacgcctt cgccgggggc gggggtacac aggcactaga 3720
cctaatccca aaacccggtg tcaaattggc tattatccaa ggcgttgcaa aacaattctt 3780
aagtaatgaa atatttttat tgacaacata tgaaaaaaat cgtataaata atattatgcg 3840
gccatggtga aatttggtaa acacatataa tttggaatta tagatacatt taagagagta 3900
tttgagggtt caagttcctc tggccgcacc atataaatct caaaatactt agcgtcgcct 3960
tcctcccggc cctttacgtc cgcctgtgaa gccctcgtcg at 4002
<210> 3
<211> 48
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tatgaccatg attacgaatt cgatatgaaa atggtgtccc tgatcgcg 48
<210> 4
<211> 48
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cttgcatgcc tgcaggtcga cgatcacgtt ctctgccttt cttcctgc 48
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gagttagctc actcattagg caccc 25
<210> 6
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gatcacgttc tctgcctttc ttcctgc 27
Claims (7)
1. one plant of bacteria cellulose produces bacterial strain, which is characterized in that the bcsB being overexpressed in acetobacter xylinum in cellulose synthase
Subunit.
2. bacteria cellulose according to claim 1 produces bacterial strain, which is characterized in that the acetobacter xylinum is acetobacter xylinum
ATCC700178。
3. bacteria cellulose according to claim 1 produces bacterial strain, which is characterized in that the bcsB in the cellulose synthase
Subunit, gene order such as SEQ ID NO:Shown in 1.
4. the construction method of any bacteria cellulose production bacterial strain of claims 1 to 3, which is characterized in that including as follows
Step：
(1) gene order of the bcsB subunits in cellulose synthase is cloned into expression plasmid, obtains recombinant plasmid；
(2) by recombinant plasmid transformed acetobacter xylinum in step (1), acetobacter xylinum gene recombination bacterium had both been obtained.
5. the construction method of bacteria cellulose production bacterial strain according to claim 4, which is characterized in that described in step (1)
Expression plasmid is pSA-19.
6. the construction method of bacteria cellulose production bacterial strain according to claim 4, which is characterized in that described in step (2)
Acetobacter xylinum acetobacter xylinum ATCC700178.
7. application of any bacteria cellulose production bacterial strain of claims 1 to 3 in bacteria cellulose is produced in fermentation.
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| WO2021110856A1 (en) | 2019-12-03 | 2021-06-10 | Crobio Ltd | Genetically engineered microorganisms |
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